Mitochondria play a major and critical role in cellular homeostasis—they participate in intracellular signaling, apoptosis and perform numerous biochemical tasks, such as pyruvate oxidation, the citric acid cycle (also referred to as the Krebs cycle), and metabolism of amino acids, fatty acids, nucleotides and steroids. However, the most crucial task of mitochondria is their role in cellular energy metabolism. This includes β-oxidation of fatty acids and production of ATP by means of the electron-transport chain and the oxidative-phosphorylation system [1, 2].
Most of the approximately 900 gene products in the mitochondria are encoded by nuclear DNA (nDNA) where mitochondrial DNA (mtDNA) only contains 13 protein encoding genes. Most of the polypeptides encoded by nDNA genes are synthesized with a mitochondrial targeting sequence (MTS), allowing their import from the cytoplasm into mitochondria through the translocation machinery (TOM/TIM). Upon entering the mitochondria, the MTS is recognized and cleaved off, allowing for proper processing and, if necessary, assembly into mitochondrial enzymatic complexes [3].
Currently, there is no cure for genetic mitochondrial metabolic disorders and treatment is mostly palliative.
Enzyme or Protein Replacement Therapy (E/PRT) is a therapeutic approach for metabolic disorders, whereby the deficient or absent protein/enzyme is artificially manufactured, purified and administered intravenously to the patient in need thereof on a regular basis.
After many years of extensive research, E/PRT has been successfully accepted as the treatment of choice for metabolic lysosomal storage diseases, including Gaucher disease, Fabry disease and attenuated variants of mucopolysaccaridoses type 1 (MPS 1). However, the inability of the intravenously administered enzymes to penetrate the blood-brain barrier severely limits the application of this approach for the treatment of other metabolic disorders that involve the central nervous system (CNS) [4, 5].
One approach for delivering proteins into cells is their fusion with protein transduction domains (PTDs), a group of short peptides that serve as delivery vectors for large molecules. Generally, PTDs are defined as short, water-soluble and partly hydrophobic, and/or polybasic peptides (at most 30-35 amino acids residues) with a net positive charge at physiological pH [6, 7]. The main feature of PTDs is that they are able to penetrate the cell membrane at low micromolar concentrations both in vitro and in vivo without using any chiral receptors and without causing significant membrane damage. Furthermore, and even more importantly, these peptides are capable of internalizing electrostatically or covalently bound biologically active cargoes (such as drugs) with high efficiency and with low toxicity. The mechanism(s) by which PTDs enter the cells has not been completely understood. One of the well-characterized PTDs is the transactivator of transcription (TAT) peptide originating from the HIV-1 virus. TAT is an 11-amino-acid (residues 47-57) arginine- and lysine-rich portion of the Tat protein encoded by HIV-1 virus [8, 9]. TAT-fusion proteins have been previously shown to be rapidly and efficiently introduced into cultured cells, intact tissue and live tissues when injected into mice [10-12]. It has also been demonstrated that TAT fusion proteins traverse mitochondrial membranes [13, WO 2009/098682].
There has been great progress in the use of PTD-fusion proteins for the delivery of different macromolecules into cells both in vitro and in vivo. This system can be used even for the delivery of cargoes into the brain across the blood-brain barrier. In addition, the ability to target specific intracellular sub-localizations, such as the nuclei, the mitochondria and lysosomes, further expands the possibilities of this delivery system to the development of sub-cellular organelle-targeted therapy. The therapeutic applications seem almost unlimited, and the use of the TAT-based delivery system has extended from proteins to a large variety of cargoes such as oligonucleotides, imaging agents, low molecular mass drugs, nanoparticles, micelles and liposomes. As will be shown, this PTD system is used for developing fusion constructs of functional mitochondrial proteins, for treatment of mitochondrial disorders, for example Friedreich ataxia.
Friedreich ataxia is an autosomal recessive degenerative disorder characterized by ataxia, areflexia, sensory loss, weakness, scoliosis, and cardiomyopathy. Diabetes mellitus, optic neuropathy, and hearing loss are also seen in patients suffering from this disease [14, 15]. Most patients with Friedreich ataxia (97%) have expansions of a GAA repeat in the first intron on both alleles of the gene encoding the mitochondrial protein frataxin [15, 16] whose expression is reduced in Friedreich ataxia [17]. The size of the GAA repeat expansion inversely correlates with frataxin expression and with the age of disease onset [16]. A deficiency of frataxin in cells leads to decreased activities of mitochondrial iron-sulfur cluster-containing enzymes, to an accumulation of iron in the mitochondrial matrix, increased sensitivity to oxidative stress, as well as to impaired adenosine triphosphate (ATP) production [18-20]. Current targets for disease-modifying drug development include agents targeting the mitochondria, aimed to (1) reduce oxidative stress and free-radical generation; (2) improve ATP production; (3) reduce iron accumulation; and (4) increase frataxin production and the assembly of iron-sulfur clusters [21]. There are presently 21 agents or classes of therapeutic agents enrolled in the research pipeline of Friedreich ataxia disease (www_curefa_org/pipeline_html). Millions of dollars from public, private, and industry-based initiatives have been dedicated to research of Friedreich ataxia therapeutics. Despite this vigorous international effort, there is as yet no proven disease-modifying therapy for Friedreich ataxia [22].
Development of E/PRT using the TAT delivery system in mitochondrial disorders was previously reported for lipoamide dehydrogenase (LAD) mitochondrial deficiency [23 and WO 2009/098682]. LAD is the E3 subunit of the three α-ketoacid dehydrogenase complexes in the mitochondrial matrix, which are crucial for the metabolism of carbohydrates and amino acids. These complexes are the pyruvate dehydrogenase complex (PDHC), the α-ketoglutarate dehydrogenase complex (KGDHC) and the branched chain ketoacid dehydrogenase complex (BCKDHC). This previously reported TAT delivery system was based on a TAT-LAD fusion protein comprising the natural precursor sequence of the human LAD containing the N-terminal 35 amino acid mitochondrial targeting sequence (MTS). The natural MTS of LAD was used to facilitate processing of the TAT-LAD construct upon delivery into the mitochondria, thus allowing the incorporation of the delivered LAD into the α-ketoacid dehydrogenase complexes. This TAT-LAD construct was demonstrated to enter patients' cells rapidly, and efficiently reaching the mitochondria. Inside the mitochondria, TAT-LAD was shown to be processed and to restore LAD activity [23]. Moreover, delivery of TAT-LAD into E3-deficient mice tissues was also demonstrated [24]. In mice tissues, a single administration of TAT-LAD resulted in a significant increase in the enzymatic activity of the mitochondrial multienzyme complex pyruvate-dehydrogenase complex within the liver, heart and, most importantly, brain of TAT-LAD-treated E3-deficient mice [24].
Notably, TAT-LAD was shown to be able to restore the activity of the pyruvate dehydrogenase complex (PDHC) within treated patients' cells almost back to its normal levels. PDHC is a 9.5×106 Da macromolecular machine whose multipart structure assembly process involves numerous different subunits: a pentagonal core of 60 units of the E2 component (dihydrolipoamide), attached to 30 tetramers of the E1 component (α2β2) (pyruvate decarboxylase), 12 dimers of the E3 (LAD, dihydrolipoamide) component and 12 units of the E3 binding protein. The structure of all α-ketoacid dehydrogenase complexes is similar to that of PDHC. The complexity of this structure emphasizes the significance in showing that TAT-mediated replacement of one mutated component restores the activity of an essential mitochondrial multi-component enzymatic complex in cells of enzyme-deficient patients.
Previous studies of mitochondria delivery system primarily used the native MTS of mitochondrial proteins (e.g. LAD) and showed that the native MTS was necessary for maximal restoration of LAD enzymatic function. Deleting the MTS restored a significantly smaller amount of LAD activity within the mitochondria. Since TAT can move both ways across a membrane and thus pull the therapeutic cargo out of the mitochondria, when MTS is included, the matrix processing peptidases recognizes the sequence and clips it, and the cargo (e.g. mature LAD) is left in the mitochondrial matrix while the TAT peptide can transduce out of the mitochondrion. Repeated dosing should therefore result in accumulating amounts of cargo in the mitochondria over time.
A TAT-FRATAXIN (TAT-FXN) fusion protein for putative treatment of Friedreich's ataxia was recently reported [25]. This TAT-FXN fusion protein was shown to bind iron in vitro, transduce into the mitochondria of Friedreich ataxia deficient fibroblasts and also reduce caspase-3 activation in response to an exogenous iron-oxidant stress. In this TAT-FXN fusion protein, the authors used the native MTS of frataxin that consists of 80 amino acid residues (aa) for preparing their TAT-FXN fusion protein [26].
It is known that FXN mRNA is translated to a precursor polypeptide that is transported to the mitochondrial matrix and processed to at least two forms, namely FXN42-210 and FXN81-210. FXN42-210 is a transient processing intermediate, whereas FXN81-210 represents the mature protein [27, 28]. However, it was found that both FXN42-210 and FXN8-210 are present in control cell lines and tissues at steady-state, and that FXN42-210 is consistently more depleted than FXN81-210 in samples from Friedreich's ataxia patients [29].
Most nuclear-encoded mitochondrial proteins contain a cleavable N-terminal MTS that directs mitochondrial targeting of the protein; as detailed above, the N-terminal MTS is cleaved off by matrix processing proteases at a well-conserved RXY↓(S/A) motif, which is a three amino acid (aa) motif, where X can be any aa, followed by serine or alanine and cleavage is performed after the three first amino acids [26, 30-31]. These N-terminal MTSs are typically 15-30 amino acids in length including 3-5 nonconsecutive basic amino acid (arginine/lysine) residues, often with several serine/threonine residues but without acidic amino acid (asparate/glutamate) residues. In their molecular structure, these MTSs are able to form strong basic amphipathic α-helices that are essential for efficient mitochondrial transportation [32]. Thus, by way of example, the long 80-aa native MTS of frataxin as well as its two-step processing can reduce its efficiency in the delivery of cargos into the matrix of the mitochondria.